There are now numerous examples of commercially valuable proteins which may be produced in larger quantities by culturing a host organism capable or expressing heterologous genetic material. Once a protein has been produced by a host organism it is usually necessary to treat the host organism in some way, in order to obtain the desired protein in a free form. In some cases, such as in the production of the interferon in E. coli a lysis or permeabilisation treatment alone may be sufficient to afford satisfactory yields. However, some proteins are produced within a host organism in the form of insoluble protein aggregates which are not susceptible to extraction by lysis or permeabilisation treatment alone. It has been reported for instance that a human insulin fusion protein produced in E. coli forms insoluble protein aggregates (see D. C. Williams et al Science Vol. 215 pages 687-689). In the normal biologically active form (hereinafter referred to as the native form) a protein exists as a chain of amino acids linked by peptide bonds. The chain is folded into a thermodynamically preferred three dimensional structure, the conformation of which is maintained by relatively weak interatomic forces such as hydrogen bonding, hydrophobic interactions and charge interactions. A number of S--S covalent bonds may form intramolecular bridges in the polypeptide chain. The insoluble proteins produced by certain host organisms do not exhibit the functional activity of their natural counterparts and are therefore in general of little use as commercial production. The lack of functional activity may be due to a number of factors but it is likely that such proteins produced by transformed host organisms are formed in a conformation which differs from that of their native states. The altered three dimensional structure of such proteins not only leads to insolubility but also diminishes or abolishes the biological activity of the protein. It is not possible to predict whether a given protein expressed by a given host organism will be soluble or insoluble.
In our copending published British patent application GB2100737A (an identical disclosure of which is contained in assignee's U.S. application Ser. No. 389,063, filed Jun. 16, 1982, now abandoned) we described a process for the production of the proteolytic enzyme chymosin. The process involves cleaving one of the chymosin precursor polypeptides; preprochymosin, methionine-prochymosin or methionine-chymosin, which may be expressed from a host organism which has been transformed with a vector includes a gene coding for the relevant protein. The process for preparing a host organism transformed with a vector carrying a suitable gene is described in detail in the specification of our published British patent application GB2100737A and the same disclosure in our U.S. application Ser. No. 389,063, filed Jun. 16, 1982, now abandoned and the teachings thereof are incorporated herein by reference.
In the course of our work relating to the process of preparing chymosin we discovered that the chymosin precursor proteins produced by various host organisms used were not produced in their native form. In particular the methionine-prochymosin produced by E. coli is almost entirely produced as an insoluble aggregate and about 75% of the methionine-prochymosin produced in Saccharomyces cerevisiae is produced in an insoluble form.
In order to produce a chymosin precursor in a native form which may be cleaved to form active native chymosin the proteins produced by a host organism must be solubilised and converted into their native form before the standard techniques of protein purification and cleavage may be applied.